The goal of proposed research is the development of novel approaches to ameliorate hypoxia-induced tissue damage. As an important mission of NIH is to develop highly innovative treatments to prevent tissue hypoxia, progression and death, the goal of the proposed work should be of high priority. Research proposed here is based on unexpected discoveries resulting from an unbiased, high-throughput metabolomic screen revealing that the metabolic pathway responsible for the production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a glycolytic erythroid byproduct known to reduce hemoglobin (Hb)-O2 binding affinity, sphingosine-1-phosphate (S1P), a highly enriched erythrocyte biolipid, and plasma adenosine, a hypoxic and energy sensor, were rapidly induced in 21 young healthy human volunteers at high altitude. Significantly, the elevation of these three metabolites was associated with the quick increase of O2 release capacity mediated by high altitude. Follow-up mouse genetic studies demonstrated that in two independent experimental models, one mimicking high altitude hypoxia and the other associated with cardiac vascular diseases, ADORA2B-mediated 2,3-BPG induction and O2 delivery has a general protective role in hypoxic tissue damage. Mechanistically, we discovered that AMPK, a known cellular energy sensor, functioned downstream of ADORA2B underlying adenosine induced 2,3-BPG production and O2 delivery. Intriguingly, we discovered that ADORA2B signaling also induces activation of erythrocyte SphK1 and that elevated SphK1-induced production of S1P functions intracellularlly to promote 2,3- BPG and O2 delivery to counteract tissue hypoxia. Functional and structural studies revealed that S1P binds Hb and synergistically works together with 2,3-BPG to further decrease Hb-O2 binding affinity and thus stabilizes deoxyHb. Overall, our published and unpublished findings raise intriguing hypotheses that elevated erythrocyte adenosine-ADORA2B-AMPK and SphK1-S1P signaling networks function collaboratively to counteract hypoxia by inducing 2,3-BPG production and O2 delivery. The project has interrelated goals to translate our findings into innovative therapeutics to treat and prevent hypoxia by providing new insight into mechanisms for hypoxia adaptation. To extend our discoveries of the protective role of these newly identified hypoxia-induced metabolites and signaling cascades, we will conduct preclinical studies (AIM I) by testing the therapeutic effects of multiple FDA approved drugs on tissue hypoxia in two independent models. AIMs II and III include pharmacological and novel genetic approaches coupled with robust innovative proteomic and metabolomic techniques to assess how AMPK and S1P-mediated hypoxic erythrocyte metabolic reprogramming to rapidly induce 2,3-BPG and O2 release. Finally, we will conduct human translational studies by collaboration with ongoing DOD funded projects to further define if AMPK and SphK1 activities are induced by high altitude hypoxia and correlated with improved cognitive function and exercise performance (AIM IV). Overall, the proposed studies are aimed to reveal important therapeutic targets in the management of high altitude and diseases associated with hypoxia.